CTxB binding has little effect on the diffusion of other cell surface molecules.

<p>COS-7 cells expressing YFP-GL-GPI or YFP-GT46, or stained with DiIC₁₆ were labeled with 1 µM Cy5-CTxB (YFP-GL-GPI and YFP-GT46) or Alexa488-CTxB (DiIC₁₆) for 5 minutes at room temperature and washed prior to FRAP studies. Diffusion coefficients were measured for YFP-GL-GPI, YFP-GT46, and DiIC₁₆ in the presence and absence of 1 µM Cy5 CTxB (mean ± SD for n = 16–32 cells). FRAP data were collected at both 20°C and 37°C. ** <i>p</i><0.01, Student t-test.</p

CTxB diffusion is confined by the actin cytoskeleton.

<p>(<b>A–D</b>) Subcellular distribution of (A) Alexa546-CTxB, (B) YFP-GL-GPI, (C) YFP-GT46 and (D) DiIC₁₆ in live COS-7 cells under control conditions or following LatA treatment as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034923#s4" target="_blank">Material and Methods</a>. A zoom of the boxed area in LatA treated cells is shown on the right for each marker. (<b>E</b>) Rhodamine phallodin staining in fixed COS-7 cells under control conditions or following LatA treatment. Bar, 10 µm. (<b>F</b>) COS-7 cells were treated with 1 µM LatA or mock-treated with 0.1% DMSO (“control”) for 5 minutes prior to imaging, and FRAP analysis was performed in the continued presence of LatA. Diffusion coefficients were measured for Alexa546-CTxB, YFP-GL-GPI, YFP-GT46 and DiIC₁₆ in control and LatA treated COS-7 cells at 37°C (mean ± SD for 13–23 cells). ** <i>p</i><0.01, Student t-test.</p

Confocal FRAP assay.

<p>COS-7 cells were transfected with the indicated constructs or left untransfected and labeled with Alexa546-CTxB or DiIC₁₆. FRAP was performed at 37°C using a 4.1 µm diameter bleach spot. (<b>A</b>) Representative images of Alexa546-CTxB during a FRAP experiment. Bar, 10 µm. (<b>B</b>) Average fluorescence intensity of Alexa546-CTxB labeling of COS-7 cells incubated with A546-CTxB concentrations ranging from 0.1 nM to 1 µM (mean ± SD for 59–151 cells). (<b>C</b>) Example of a normalized recovery curve for a cell labeled with 1 µM Alexa546-CTxB after correcting for fluorescence decay during imaging, along with fitted FRAP curve. (<b>D</b>) Representative example of <i>D</i> for CTxB as a function of time after labeling. Each value of <i>D</i> was obtained for a different cell on the same coverslip from a single experiment. (<b>E</b>) Representative whole cell images of YFP-GT46, YFP-GL-GPI, and DiIC₁₆ in COS-7 cells. Single confocal slices are shown. The spotty appearance of DiIC₁₆ on the background is due to the presence of dye aggregates. Bar, 10 µm. (<b>F</b>) Representative FRAP curves for Alexa546-CTxB, YFP-GT46, YFP-GL-GPI, and DiIC₁₆ (n = 8–13 cells for each).</p

CTxB diffusion is confined by ATP-dependent barriers.

<p>(<b>A–E</b>) Subcellular distribution of Alexa555-CTxB and YFP-GL-GPI in control and ATP-depleted COS-7 cells. Images show the projection of a series of confocal slices through live cells. Arrows mark the position of an xz-section (shown below.) Scale bar = 10 µm. (A, D) Typical morphology of cells labeled with Alexa555-CTxB or expressing YFP-GL-GPI under control conditions. (B, E) In ATP depleted cells, in addition to labeling the bulk of the plasma membrane, Alexa555-CTxB and YFP-GL-GPI label protrusions of the plasma membrane found close to the coverslip, as well as protrusions projecting above the surface of the cells into the media. (C) Example of an ATP depleted cell in which CTxB accumulates in tubular plasma membrane invaginations in addition to protrusions. (<b>F</b>) COS-7 cells were ATP depleted or mock-depleted (“control”) for 15 minutes prior to labeling and FRAP was performed in the continued presence of ATP depletion or control medium. Diffusion coefficients were measured for Alexa546-CTxB, YFP-GT46, YFP-GL-GPI or DiIC₁₆ (mean ± SD from 24–32 cells). Cells were labeled with 1 µM Alexa546-CTxB. FRAP was performed at 37°C. ** <i>p</i><0.01, Student t-test.</p

ATP depletion induces actin polymerization.

<p>Rhodamine-phalloidin labeling in mock-depleted and ATP depleted COS-7 cells (<b>A</b>) labeled with Alexa488-CTxB or (<b>B</b>) expressing YFP-GL-GPI. A single confocal section is shown for each. A zoom of the boxed area is shown on the right. The merged images show phalloidin staining in red and CTxB or YFL-GL-GPI staining in green. The spotty appearance of CTxB and YFP-GL-GPI is the result of fixation and permeabilization conditions. Scale bar = 10 µm.</p

Caveolae have little effect on CTxB diffusion at the cell surface.

<p>(<b>A</b>) Subcellular distribution of Alexa546-CTxB, YFP-GL-GPI, YFP-GT46, and DiIC₁₆ in live Cav-1^+/+ and Cav-1^−/− MEF cells. Bar, 10 µm. (<b>B</b>) Diffusion coefficients of Alexa546-CTxB, YFP-GL-GPI, YFP-GT46 and DiIC₁₆ in Cav-1^+/+ and Cav-1^−/− MEF cells (mean ± SD from 22–47 cells). Cells were labeled with 1 µM Alexa546-CTxB. FRAP was performed at 37°C.</p

Representative photofading rates obtained from the whole image fluorescence for a variety of different fluorescently tagged molecules.

<p>A photofading model, <i>f</i>(<i>t</i>) = <i>e</i>^{−<i>κt</i>} was fitted to normalized whole image fluorescence data <i>F</i>_<i>Data</i>(<i>t</i>)/<i>F</i>_<i>i</i> averaged over multiple data sets (<i>n</i> = 10) for each of the indicated fluorescent proteins or fluorescent lipid probes. (A–B) Best fitting photofading model applied to whole image fluorescence. Solid lines show the best fitting curves. (C) Photofading rate constants obtained from the fits shown in A and B are shown in descending order. Error bar represents the standard deviation (<i>n</i> = 14).</p

Errors caused by incorrect sequence of scaling or by applying partial scaling corrections.

<p>FRAP analysis was performed on Alexa488-CTxB FRAP data (A–D) or FLOT-RFP FRAP data (E–H) using different scaling scenarios. (A,E) As a positive control, FRAP data corrected for both background fluorescence and photofading were analyzed using <i>f</i>_{<i>NDaM</i>_<i>f</i>}(<i>t</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127966#pone.0127966.e052" target="_blank">Eq 37</a>) in the correct order. (B,F) Background-corrected FRAP data were analyzed by <i>f</i>_{<i>NDaM</i>_<i>f</i>}(<i>t</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127966#pone.0127966.e052" target="_blank">Eq 37</a>), ignoring the contribution of photofading. (C,G) Photofading-corrected FRAP data, <i>F</i>_<i>Data</i>(<i>t</i>)/<i>F</i>_<i>Whole</i>(<i>t</i>) were analyzed by <i>f</i>_{<i>NDaM</i>_<i>f</i>}(<i>t</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127966#pone.0127966.e052" target="_blank">Eq 37</a>), ignoring any background correction. Lower lines show background fluorescence, <i>F</i>_<i>b</i>. (D,H) Background corrected FRAP data were analyzed using a photofading and mobile fraction corrected FRAP equation obtained by using an incorrect scaling order (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127966#pone.0127966.e059" target="_blank">Eq 44</a>). Dots (•) show the mean FRAP data averaged over one set of experiments (<i>n</i> = 12 and 13 for FLOT-RFP and Alexa488-CTxB, respectively) and solid lines show the best fitting curve.</p

Depletion of PACSIN2 inhibits TcdA entry in Caco-2 cells.

<p><b>(A)</b> Caco-2 cells expressing non-targeting shRNA (Ctrl shRNA) or shRNA 982 targeting PACSIN2 were incubated with 50 nM TcdA-546 at 10°C for 45 min. Unbound toxins were removed and cells were shifted to 37°C to allow internalization. After 20 min, cells were washed, fixed, stained for PACSIN2 and imaged by confocal microscopy. PACSIN2 and TcdA staining from ctrl shRNA and sh982 expressing cells are shown. Scale bars, 10 μm. The images shown are representative of multiple fields imaged from three independent experiments <b>(B)</b> Scatter plot of the relative fluorescence intensities of PACSIN2 and TcdA in ctrl shRNA (black circles) and PACSIN2 sh982 (white squares) expressing cells. Each data point represents an individual cell. A total of 78 cells per condition were chosen at random for analyses. Linear regression analysis was performed in GraphPad Prism and indicates a strong correlation between PACSIN2 and toxin levels in cells. <b>(C)</b> Comparison of mean fluorescence intensities of PACSIN2 between ctrl shRNA and PACSIN2 sh982 expressing cells. Data represent mean and SD of 78 individual cells. Student’s t test ***p<0.0001. <b>(D)</b> Comparison of mean fluorescence intensities of TcdA between ctrl shRNA and PACSIN2 sh982 expressing cells. Data represent mean and SEM of 78 individual cells. Student’s t test ***p<0.0001.</p

Effect of photofading during image acquisition on fits to FRAP curves for normal diffusion versus anomalous diffusion.

<p>FRAP data with photofading (•) were simulated using <i>f</i>_{<i>dADaM</i>_<i>f</i>}(<i>t</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127966#pone.0127966.e057" target="_blank">Eq 42</a>) assuming either a photofading rate of <i>κ</i> = 0.04 s⁻¹ (A,B) or <i>κ</i> = 0.01 s⁻¹ (C,D) under the conditions <i>D</i> = 1<i>μ</i>m²/s, <i>F</i>₀ = 0.2, <i>M</i>_<i>f</i> = 0.8, <i>r</i>_<i>n</i> = 0.2 <i>μ</i>m, and <i>r</i>_<i>e</i> = 1 <i>μ</i>m. The simulated curve was next fitted by <i>f</i>_{<i>NDaM</i>_<i>f</i>}(<i>t</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127966#pone.0127966.e052" target="_blank">Eq 37</a>) (A,C) or <i>f</i>_{<i>NDaM</i>_<i>f</i>}(<i>t</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127966#pone.0127966.e052" target="_blank">Eq 37</a>) (B,D). The resulting best fitting parameters were <i>D</i> = 2.1<i>μ</i>m²/s and <i>M</i>_<i>f</i> = 0.83 in (A), </p><p></p><p></p><p><mn>1</mn><mn>4</mn></p><mo>Γ</mo><mo>=</mo><mn>9</mn><mo>.</mo><mn>33</mn><p></p><p></p> and <i>M</i>_<i>f</i> = 0.83 in (B), <i>D</i> = 2.7<i>μ</i>m²/s and <i>M</i>_<i>f</i> = 0.95 in (C), and <p></p><p></p><p><mn>1</mn><mn>4</mn></p><mo>Γ</mo><mo>=</mo><mn>5</mn><mo>.</mo><mn>32</mn><p></p><p></p> and <i>M</i>_<i>f</i> = 0.94 in (D).<p></p